FORMULATION DE LIPIDES IMMUNOSTIMULANTS

IMMUNSTIMULATING LIPID FORMULATION

Abrégé français

Formulation pharmaceutique servant à administrer par voie parentérale ou muqueuse des antigènes ou des vaccins à l'homme et à l'animal et contenant des préparations de monoglycérides possédant au moins 80 % de monoglycérides et dans lesquels le groupe acyle contient de 6 à 24 atomes de carbone, ainsi que des acides gras, le nombre des atomes de carbone pouvant varier entre 4 et 22.

Abrégé anglais

A pharmaceutical formulation for parenteral or mucosal administration of
antigens and/or vaccines to humans and animals, comprising monoglyceride
preparations having at least 80% monoglyceride content and where the acyl
group contains from 6 to 24 carbon atoms, together with fatty acids where the
number of carbon atoms may be varied between 4 and 22.

Revendications

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CLAIMS:
1. An adjuvant for use in a vaccine for parenteral or
mucosal administration of an antigen to an animal,
comprising
i) one or more monoglycerides each having a purity of at
least 80%, the one or more monoglycerides independently
having the general formula
<IMG>
wherein R1, R2 and R3 are selected from H and an acyl chain
comprising from 6 to 24 carbon atoms, and wherein the acyl
chain optionally comprises one or more unsaturated bonds,
with the proviso that two of the groups R1, R2 and R3 are H;
ii) one or more fatty acids comprising 6 to 24 carbon atoms,
and where the acyl chain thereof optionally contains one or
more unsaturated bonds; and
iii) water;
wherein the concentration of i) is from 0.1 g to 50 g per
100 ml of water, and the concentration of ii) is
from 1 g to 20 g per 100 ml of water.
2. An adjuvant according to claim 1, wherein the
purity of each of the one or more monoglycerides is at
least 90%.
3. An adjuvant according to claim 1, wherein the
purity of each of the one or more monoglycerides is at
least 95%.

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4. An adjuvant according to any one of claims 1 to 3,
wherein the number of carbon atoms in the acyl chain of R1,
R2 or R3 is 8 to 20.
5. An adjuvant according to any one of claims 1 to 3,
wherein the number of carbon atoms in the acyl chain of R1,
R2 or R3 is 14 to 20.
6. An adjuvant according to any one of claims 1 to 5,
wherein the number of carbon atoms in the acyl chain of the
one or more fatty acids is 8 to 20.
7. An adjuvant according to any one of claims 1 to 5,
wherein the number of carbon atoms in the acyl chain of the
one or more fatty acids is 14 to 20.
8. A vaccine composition comprising an adjuvant
according to any one of claims 1 to 7, and an immunogenic
quantity of an antigen component.
9. A vaccine composition according to claim 8,
wherein the antigen component is for causing an immune
response in an animal.
10. A vaccine composition according to claim 9,
wherein the animal is a human or a marine animal.
11. A vaccine composition according to any one of
claims 8 to 10, wherein the antigen component is selected
from the group consisting of antigens from pathogenic and
non-pathogenic bacteria, viruses, parasites and tumor cells.
12. A vaccine composition according to any one of
claims 8 to 11, wherein the adjuvant further comprises one
or more pharmaceutically acceptable excipients selected from
the group consisting of: preservatives, osmotic pressure
controlling agents, pH-controlling agents, organic solvents,

-16-
hydrophobic agents, enzyme inhibitors, water absorbing
polymers, surfactants, absorption promoters, and anti-
oxidative agents.
13. A vaccine composition according to any one of
claims 8 to 12, wherein the adjuvant is for administration
to the mucosa of the nose, mouth, vagina, rectum or the
intestine.
14. A vaccine composition according to any one of
claims 8 to 12, wherein the composition is for
administration to the mucosa of the nose.
15. A vaccine composition according to any one of
claims 8 to 14, comprising per 100 g of the composition:
from 0.01 to 90 g of the antigen component;
from 0.1 to 50 g of the one or more monoglycerides; and
from 1 to 20 g of the one or more fatty acids;
from 0.01 to 99 g of water; and
from 0.01 to 99 g of PBS/saline.
16. Use of an adjuvant as defined in any one of
claims 1 to 7 in production of a pharmaceutical composition
for enhancing immune response to an antigen in an animal.
17. Use of a vaccine composition as defined in any one
of claims 8 to 15 in production of a pharmaceutical
composition for immunizing an animal.

Description

CA 02258017 1998-12-09
WO 97/47320 PCT/SE97/01003
IMMUNSTIMULATING LIPID FORMULATION
The present invention relates to a novel pharmaceutical formulation for
administration
of antigens and/or vaccines. The preferred route of administration is via the
mucosal
membranes, however parenteral administration may also be used. The invention
also
relates to the use of certain compounds (as defined below) as adjuvants or
vehicles in such
formulation.
Background
An increasing number of specific antigens from different types of organisms
(e.g.
tumor cells, bacteria, virus and parasites) has been produced using cloning
techniques over
the last years. However, these antigens are frequently weak immunogens despite
their high
specificity.
To obtain good protection after vaccination, immune stimulating systems are
needed
that can enhance and activate the immune system against these weak antigens.
Such
immune stimulating systems are called adjuvants.
Adjuvants, presently mainly used in animal experiments, includes a highly
heterogeneous group of substances; inorganic substances, oil emulsions,
charged
polymers, neutral substances or substances from bacteria.
There are presently large efforts in research and development in order to
obtain a safe
adjuvant with high efficacy to be used in humans. However, today there is
presently no
general adjuvant for this purpose.
Alum hydroxides and alum phosphates were the first two inorganic substances
that
were used in humans. The immune response obtained is a result of slow
desorption of the
precipitated antigen on the surface of the particle. Later it was shown that
phagocyting
cells were attracted by these alum salts leading to further enhancement of the
immune
response. However, these salts are not safe since granuloma formation has been
reported
(Slater et al, Br.J.Dermatol. (1982) Vol. 107, page. 103-108.). Furthermore,
the alum salts
can not be used for all antigens since all antigens are not adsorb on the
surface.
In 1944 Freund introduced his adjuvant consisting of a mixture of vegetable
oil,
mineral oil, detergents and killed bacteria. The enhancement obtained was
partly due to
slow release of the antigen from the oil emulsion. Freunds adjuvant can
however not be
used in humans due to granuloma formation, induction of auto-immune reactions
and the
non-biodegradable mineral oil. Furthermore, the effect is difficult to
control. The active

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substance in Freunds adjuvant has been isolated and its structure determined
and shown to
be N-acetyl muramyl-L-alaninisoglutamate, often called muramyl-dipeptide
(MDP).
The adjuvant effect dependent of the particle size of polymetacrylate and
polystyrene
particles was examined on mice (Kreuter et al, Vaccine, (1986) vol 4, 125-129)
by the use
of ovalbumin (adsorbed on the particles) as a model antigen with subsequent
assay of the
immune response. The size of the particles was varied between 62 and 306 nm.
The result
was that smaller particles enhanced the immune response better than larger.
The smaller
particles gave a better effect than 0.2% Al(OH)3 . All preparations elicited a
higher
response as compared to fluid preparations. Similar experiments where
particulate systems
with smaller size results in a higher immune response as compared to larger
particles are
known in the scientific literature.
Almost all systems used today for enhancement of the immune response against
antigens are particles or is forming particles together with the antigen. In
the book "Vaccine
Design - the subunit and adjuvant approach" (Ed: Powell & Newman, Plenum
Press,
1995) all known adjuvants are described both regarding their immunological
activity as
well as regarding their chemical characteristics. As described in the book
more than 80%
of the adjuvants tested today are particles or polymers that together with the
antigens (in
most cases proteins) are forming particles. The type of adjuvants that not are
forming
particles are a group of substances that are acting as immunological signal
substances and
which under normal conditions consists of the substances that are formed by
the immune
system as a consequence of the immunological activation after administration
of particulate
adjuvant systems.
Using particulate systems as adjuvants, the antigens are associated or mixed
with or to
a matrix which has the characteristics of being slowly biodegradable. Of great
importance
using such matrix systems are that the matrix does not form toxic metabolites.
Choosing
from this point of view, the main kind of matrices that can be used are mainly
substances
originating from a body. With this background there are only a few systems
available that
fulfils these demands: lactic acid polymers, poly-amino acids (proteins),
carbohydrates,
lipids and biocompatible polymers with low toxicity. Combinations of these
groups of
substances originating from a body or combinations of substances originating
from a body
and biocompatible polymers can also be used. Lipids are the preferred
substances since
they display structures that make them biodegradable as well as the fact that
they are the
most important part in all biological membranes.
_.T

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Lipids are characterized as polar or non-polar. The lipids that are of most
importance
in the present invention are the polar lipids since they have the capacity to
form particulate
systems in water. Another way of defining these lipids are as amphifilic due
to their
chemical structure with one hydrophobic and one hydrophilic part in the
molecule thereby
being useable as surface active substances. Examples of main groups of polar
lipids are
mono-glycerides, fatty acids, phospholipids and glycosphingolipids. These main
groups
can be further characterized depending on the length of the acyl chain and the
degree of
saturation of the acyl chain. Since the number of carbon atoms in the acyl
chain can be in
the range of 6 to 24 and the number of unsaturated bonds can be varied there
are an almost
infinite number of combinations regarding the chemical composition of the
lipid.
Particulate lipid systems can be further divided into the different groups as
discussed
in the scientific literature such as liposomes, emulsions, cubosomes,
cochleates, micelles
and the like.
In a number of systems the lipids may spontaneously form, or can be forced to
form,
stabile systems. However, under certain circumstances other surface active
substances has
to be introduced in order to achieve stability. Such surface active systems
can be of non-
lipid character but possess the characteristics of the polar lipids having
hydrophobic and
hydrophilic parts in their molecular structure.
Another factor that has been shown to be of importance is that lipids exhibit
different
physical chemical phases, these phases has in different test systems been
shown to
enhance uptake of biological substances after administration to mucosal
membranes.
In the classical immunology and in combination with vaccination against
different
types of infectious agents e.g. bacteria, virus or parasites the prevailing
dogma has been to
administrate the vaccine subcutanously or intramuscularly. However, research
has during
the last years shown that the body has a very effective immunological system
that resides
in the mucosa. It has been shown that you can administrate vaccines orally,
nasally, rectally
and vaginally. In the same way as for the classical immunization it has been
shown that by
mucosal vaccination there is also a need for enhancement of the immunological
response
by the addition of adjuvants.
In the same way as within the classical immunology where vaccines (antigens)
are
administrated parenterally, there is within mucosal immunization a great
interest in
directing the immunological response towards development of humoral and/or
cellular
response. If you obtain a humoral response it would be important to direct the
response in
a way that a certain class of antibodies would be obtained. In order to obtain
such a goal,

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specific immune stimulating agents can be added to the formulation of antigens
and
adjuvants.
Different types of immune stimulating substances are available. One type is
represented by proteins e.g. PHA, Con A, SEA or different types of interferons
or
interleukines. Another type of substance is represented by MDP, as mentioned
above.
Additional groups can be characterized as lipid derivatives since they show
molecular
structures which are amphiphilic. One example of such a substance is called
MPL.
Another similar substance is Quil A. A number of substances that can be
classified within
these categories are described in the book "Vaccine Design - the subunit and
adjuvant
approach" as discussed above.
It would be extremely valuable to be able to make the immunization procedures
more
effective directing the immunological response towards a certain class or
subclass of
antibodies and/or to be able to induce a strong T-cell response against the
antigens.
Description of the invention
It has now surprisingly been found that parenteral or mucosal administration
of a
pharmaceutical formulation containing one or two of the following adjuvants
with admixed
antigens and/or vaccines improves the immune response against the admixed
antigens/vaccines. Said pharmaceutical formulation for parenteral or mucosal
administration of antigens and/or vaccines to an animal comprise one or more
substances
selected from
a) monoglycerides of the general formula
CH2-CH -CH2
0 0 0
1 1 1
R1 R2 R3
wherein R, and R2 is H and R3 is one acyl group containing from 6 to 24 carbon
atoms, preferably 8 to 20 carbon atoms, even more preferably 14 - 20 carbon
atoms and where the acyl chain may contain unsaturated bonds. In a
monoglyceride the acyl chain is normally in the R, or R3 Position. However
there is
normally a acyl migration between the 1 and 2 carbons in the glycerol molecule
resulting in approximately 90 % is in the R3 position and 10% in the R2
position.
Thus, in the present invention distilled I -monoglycerides from Danisco
Ingredients
(Denmark) with a purity of more than 80 % preferably more than 90 %, more

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preferably over 95% is used. The diglyceride content is
maximum 3% and triglycerides and fatty acid content is less
than 1.0%. The monoglycerides according to the invention
normally contains more than 80% of a specific fatty acid,
preferably over 90%;
and
b) fatty acids of the general formula
CH3- (CH2) n-COOH
where "n" may be varied between 4 and 22,
preferably 8 to 18 and where the acyl chain may contain one
or more unsaturated bonds.
According to one aspect of the present invention,
there is provided an adjuvant for use in a vaccine for
parenteral or mucosal administration of an antigen to an
animal, comprising
i) one or more monoglycerides each having a purity of at
least 80%, the one or more monoglycerides independently
having the general formula
CH2-?H-?H2
O IIO 0
1 1
R1 R2 R3
wherein R1, R2 and R3 are selected from H and an acyl chain
comprising from 6 to 24 carbon atoms, and wherein the acyl
chain optionally comprises one or more unsaturated bonds,
with the proviso that two of the groups R1, R2 and R3 are H;
ii) one or more fatty acids comprising 6 to 24 carbon atoms,
and where the acyl chain thereof optionally contains one or
more unsaturated bonds; and

CA 02258017 2010-06-02
22903-969
-5a-
iii) water;
wherein the concentration of i) is from 0.1 g to 50 g per
100 ml of water, and the concentration of ii) is
from 1 g to 20 g per 100 ml of water.
According to another aspect of the present
invention, there is provided a vaccine composition
comprising an adjuvant as described herein and an
immunogenic quantity of an antigen component.
According to still another aspect of the present
invention, there is provided use of an adjuvant as described
herein in production of a pharmaceutical composition for
enhancing immune response to an antigen in an animal.
According to yet another aspect of the present
invention, there is provided use of a vaccine composition as
described herein in production of a pharmaceutical
composition for immunizing an animal.

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22903-969
5b
The formulation according to the invention may comprise additional
pharmaceutical
excipients selected from the one or several of the following groups;
preservatives and
osmotic pressure controlling agents, pH-controlling agents, organic solvents,
hydrophobic
agents, enzyme inhibitors, water absorbing polymers, surfactants and
absorption
promoters, anti-oxidative agents, and the like.
The formulation according to the invention may comprise any antigen and/or
vaccine
selected among all the antigen and/or vaccines relevant to humans or animals,
including
marine animals.
This invention discuss lipids which, when mixed with antigens, enhance the
immune
activity against the antigens thereby functioning as an adjuvant in various
vaccine
formulations. Especially the invention comprise the use of a formulation for
vaccination of
the mucosa which can be immunologically activated by nasal, oral, vaginal or
rectal
administration. The invention also comprise the use of the lipid system for
parenteral .
administration. The use of an 'adjuvant such as described in the present
invention, which
can be used both for parenteral as well as for mucosal administration is not
limited to
humans. Equally important is the use within the veterinary field for the
immunization of
e.g. cattle, pigs, chickens and the like. Furthermore, there is a large and
growing interest in
applying both parenteral as well as mucosal vaccines in the field of fish
farming. In this
area the administration can be performed by incorporation of the formulation
in the food.
Furthermore, the fish may be allowed to swim for a limited period of time in
the vaccine
formulation containing the antigens and the adjuvants thus being immunized by
the
mucosal route via the gills.
In the scientific literature there are reports showing how to enhance the
uptake of a
biologically active substance after administration to the mucosa together with
certain lipids.
As an example Li & Mitra (Pharm.Res. vol 13:1, 1996) describes the
administration of

CA 02258017 1998-12-09
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insulin mixed with phospholipids in the form of liposomes to the lung. They
show that the
effect is dependent on the length of the acyl chain and the charge of the
particle. Optimal
length was 10 carbon atoms and the charge preferably positive. Even negatively
charged
particles were effective but neutral system were inferior.
In the same way de Haan et al (Vaccine, 13:2, 155-62, 1995) describes a
mixture of
liposomes and the antigen hemeagglutinin. The mixture was administrated
nasally to rats
whereafter a positive immunological response could be detected. Gupta et al
(Vaccine,
14:3, 219-25, 1995) describes that a mixture of diphtheria toxoid together
with a non-
phospholipid based liposom system administrated parenterally to rabbits
results in an
immune response which was at the same level as the marketed product which was
Alum-
adsorbed diphtheria toxoid.
A number of scientific reports also show that good immunological reponses are
obtained after administration of liposomes to the mucosa where the antigen is
entrapped or
adsorbed to liposomes.
Studies in vitro on a human cell line obtained from a colon cancer (Caco-2)
shows that
the best penetrating effect, tested with the model substance mannitol, can be
seen with a
chain length of 10 carbon atoms. In this case the lipids consisted of the
salts of fatty acids.
The obtained mixture of these lipids forms together with water micelles
(Lindmark et al,
J.Pharm.Exp.Ther. 275, 958-65, 1995).
Liposomes consists of phospholipids and are formulated by a relatively lengthy
and
cumbersome process which i.a. involves organic solvents. Furthermore, the
phospholipids
are expensive.
As described below in the present invention, a similar immunological response
can be
obtained only by mixing the antigen with a lipid formulation which contains
less
complicated lipids having a substantially lower price and which can be
formulated on a
commercial basis in a very simple way.
Another systems that to some extent are similar to the present invention are
formulations based on triglycerides. However, these systems are scientifically
defined as
emulsions of triglycerides where surfactants are used for stabilization. As
stabilizers
phospholipids or any other type of amphiphilic molecules such as Tween are
normally
used. Furthermore, the appearance of such emulsions are normally milky,
indicating a size
of the oil droplets of about 1 m. It is well-known for the person skilled in
the art that these
surfactants are excellent adjuvants. Thus, the adjuvant properties of oil
emulsions are
primarily due to the characteristics of the surfactant and not of the
triglyceride composition.

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In WO 97/017827 is disclosed a formulation for the topical administration of
antigens and/or vaccines to mammals via the mucosal membranes. Said
application
disclose in the examples that the only formulation that enhances the immune
response is a
combination of caprylic/capric acid glycerides with polyoxyethylene sorbitan
monoester
(Tween 20'*1).
As exemplified in the present invention it is shown that a combination between
a
monoglyceride and a fatty acid can stimulate the immune system to produce
antibodies and
induce protective immunity. Furthermore the present invention shows that the
disclosed
formulation is able to produce high antibody titers by parenteral
administration.
10, Thus, it was surprisingly found that the administration of antigens and/or
vaccines to
an animal either via the mucosal route or parenterally using a formulation
comprising
monoglycerides and/or fatty acids as a particulate lipid system can improve
the
immunological response towards the admixed antigens andior vaccines. The
monoglycerides are selected from a group with the general formula of I -acyl-
glyceride,
wherein the number of carbon in the acyl chain may be varied between 8 and 24,
preferably between 12 and 18. The acyl chain may be either saturated or
unsaturated. The
concentration of the monoglyceride may be in the range of 0.1 - 50 g per 100
ml of water,
preferably in the range of 1- 20 g per 100 ml of water. The fatty acid
concentration may be
in the range of 0.1 - 50 g per 100 ml formulation, preferably in the range of
1 - 20 g per
100 ml water. When monoglycerides and fatty acids are formulated together the
percent
ratio of monoglyceride in fatty acid may be varied between 1 to 99 %,
preferably between
10 to 90%.
An enhancement of the immunological response after administration of
monoglycerides and/or fatty acids together with antigens and/or vaccines has
not been
suggested anywhere in the prior art.-
The present invention describes that mixtures of antigens with relevant lipids
stimulates the body to generate protective immunity. Another advantage of the
present
invention is the simple formulation process and as compared to entrapment no
material
(antigen) is lost in the process. As an example can be mentioned that in the
process of
entrapment in liposomes the recovery is normally 10-20 %. The rest is lost in
the process.
Reports in the literature as discussed above, shows that by mixing liposomes
and
antigen an immune response is detected after administration to the mucosa.

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However, the examples in this invention as described below shows that the
system
can be even more simplified by the use of lipids that are more stable, cheaper
and which
can be formulated to particles in a more convenient and simplified way.
The invention is exemplified by the following examples showing that the
principle of
co-administration oi' antigens, immune stimulating substances associated or in
combination with particles function as an adjuvant.
Example 1.
A suspension of mono-olein was produced by adding 3 g mono-olein to 50 ml of a
0.6 % Pluronic-127 solution in phosphate buffered saline pH 7.4, whereafter
the mixture
was sonicated with a probesonicator for 4 minutes. The obtained milky
suspension
contained particles with a maximal size of about 2 m as determined by light
microscopy.
Example 2.
A negatively charged micelle suspension of mono-oleate was produced by mixing
of
0.5 g of oleic acid with 5 ml of 0.35 M NaOH and sonicated with a
probesonicator for 5
seconds. Thereafter 3 g mono-olein and 50 ml 0.9 % NaCl was added whereafter
the
mixture was probesonicated for 4 minutes. The monester content of the mono-
oleate was
over 95 % with a acyl chain containing 92 % oleate and 6% linoleic acid. The
pH was
adjusted to 8.3. The obtained completely clear homogenous solution contained
particles
with a size of below approximately 0.2 m as determined by visual inspection.
It is known
that if a clear solution is obtained the particle size is below approximately
0.2 m, a slightly
opalescent bluish appearance indicated a size of approximately 0.2 - 0.5 pm
and if the
appearance is milky the size is above approximately 0.8 m.
Example 3.
A positively charged micelle suspension of mono-olein was produced by mixing
0.5
g lauryl-amine and 3.5 ml of 0.5 M HCl followed by sonication for 5 seconds.
Thereafter
3 g mono-olein and 50 ml of water was added whereafter the mixture was
probesonicated
for 4 minutes. The pH was adjusted to between 4 and 5 using 0.5 M HCI. The
obtained
completely clear homogenous solution contained particles with a size of below
approximately 0.2 gm.

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Example 4.
A mixture of particles according to Example I and diphtheria toxoid was
administrated subcutanously to mice followed by a booster after 21 days. After
30 days
blood samples were obtained which were assayed for IgG antibodies against
diphtheria
toxin as well as Neutralization titers (NT) using Vero cells. The serum from
Alum (n=5)
and monoolein (n=5) groups was pooled and assayed. The mice receiving nasal
boost
and responded (= 3 of 5) were assayed on an individual basis. In arbitrary
units is shown
in Table I the IgG titers and neutralization titers The results showed that
both IgG as well
as protective antibody titers were at the same level as compared to the
control group which
received the marketed product comprising diphtheria toxoid adsorbed on Alum
(AI(PO4)3).
Also seen is that high IgG titers always were accompanied by high
neutralization titers
indicating that the formulation does not destroy the antigenic sites that are
important for
protective immunity.
Table 1.
Dose diphtheria toxoid g IgG titer NT titer
(arb.units) (arb.units)
Alum 15 + 15 32000 40000
Alum 3.5+3.5 22000 20000
Mono-olein suspension 15 + 15 24000 20000
Mono-olein suspension 3.5+3.5 3500 5000
Nasal boost 7+4 45000 10000
Nasal boost 7+4 19000 2500
Nasal boost 7+4 19500 5000
Example 5.
Particles were prepared according to Example 2 with a final concentration of
monoglyceride of 200 mM and of fatty acid of 200 mM. Diphtheria toxoid (2.9
l, 4.4
mg/ml) was mixed with 200 l of the micelle suspension and administrated
subcutanously
to mice followed by a subcutaneous booster after 21 days. Both the primary and
the
booster dose of the toxoid was 10 g. After 30 days blood samples were
obtained which
were assayed for IgG antibodies against diphtheria toxin. The result showed
(Table 2) that
the arbitrary IgG titers with respect to the formulation with mono-olein (MO)
and oleic
acid (C 18:1) were at the same level as compared to the control group which
received the

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present marketed product comprising diphtheria toxoid adsorbed on Alum
(A1(PO4)3). The
other combinations of monoglycerides and fatty acids gave slightly declining
responses
which correlated to declining length of the acyl chain (M 12 = lauryl- l -
glycerate; M 10 =
capric-l -glycerate; ('12 = lauric acid; CIO = capric acid; C8 = caprylic
acid). N.D. = Not
Done; indicates that there were only five mice in these groups.
Table 2.
IgG response of individual mice (n = 5 or 6) after sc/ sc administration of
different
formulations containing monoglycerides and fatty acids.
1 2 3 4 5 6
Alum 18200 18200 9600 9600 18200 N.D.
MO / C18:1 18200 18200 18200 18200 9600 N.D.
MO / C8 9600 9600 4800 9600 4800 9600
M12/C12 18200 9600 4800 18200 9600 18200
M 10 / C 10 4800 110 2400 1200 2400 4800
Example 6.
The same procedure as in Example 4 with the difference that the booster dose
was given
nasally instead of subcutanously. The dose of diphtheria toxoid was 10 p.g
both at the
primary immunization as well as at the nasal booster administration. In the
same
experiment a dose-response is demonstrated that is obtained when tree
different amounts
of lipid (see Table 3) was administrated. The arbitrary IgG titer is seen in
Table 4. Besides
the dose-response effect where lower IgG titers is seen at lower
concentrations of lipids
there is also seen a higher variability regarding response in the groups
receiving lower
doses. This variability is not seen at higher dose levels indicating that an
adjuvant effect is
not only seen with respect to obtaining high titers but also regarding
reduction of the
variability of the response.
Table 3.
Amount of lipids in pmol administrated to mice sc or nasally.
Dose level Dose lipid (gmol) sc Dose lipid (pmol) nasally
high 40 1.5
medium 4 0.15
low 0.4 0.015
T

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PCT/SE97/01003
Table 4.
IgG titers in individual mice (n = 6) after administration of 2 x 10 g of
diphtheria
toxoid to mice either sc/sc or sc/nasally.
1 2 3 4 5 6
MO / C8 sc/sc high 4800 4800 9600 4800 9600 9600
MO / C8 sc/nas high 9600 1200 4800 4800 4800 9600
MO / C8 sc/sc medium 4800 1200 9600 2400 4800 4800
MO / C8 sc/nas medium 2400 600 2400 600 2400 4800
MO / C8 sc/sc low 300 2400 9600 2400 4800 2400
MO / C8 sc/nas low 600 600 1200 150 4800 150
Example 7.
Two different lipid formulation containing mainly medium length acyl chains
(Composition A) and long acyl chains (Composition B) were tested. The
compositions are
seen in Table 5.
Table 5.
Monoglyceride Fatty acid
Composition A Monooleate 25 mM Caprylic acid 90 mM
Monomyristate 25 mM
Monolaurate 25 mM
Monocaprate 25 mM
Composition B Monooleate 200 mM Oleic acid 200 mM
The formulations were administrated to mice s.c. or nasally with a booster
after three
weeks s.c. or nasally. Blood samples were taken after another week. The
arbitrary IgG
titers are seen in Table 6.
The results in Table 6 demonstrates that in order to achieve a good response
after
primary as well as booster administration by the nasal route Compositions B is
to be
preferred.

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Table 6.
1 2 3 4 5 6
Composition A sc/nas 2400 4800 4800 18200 4800 4800
Composition A nas/nas < 100 36400 < 100 < 100 300 < 100
Composition B sc/nas 18200 18200 36400 18200 < 100 N.D.
Composition B nas/nas 4800 9600 18200 18200 2400 9600
Example 8.
A mixture of mono-olein (200 mM) and caprylic acid (200 raM) was mixed with
formalin inactivated influenza virus (strain SDA/94) and administrated s.c. at
the first
occasion to mice followed by a nasal booster three weeks later. The dose was
0.05 gg HA
and blood sample were taken 3 weeks after the booster dose and assayed for
agglutination
titers (HI) against HA. The results (Table 7) showed that the HI titers in the
group
receiving the virus together with the adjuvants was at a higher level as
compared to the
group receiving the virus in PBS.
Table 7.
HI titers in mice receiving formalin inactivated influenza virus after s.c.
primary
injection and nasal booster.
1 2 3 4 5 6
PBS N.D. 80 N.D. 40 N.D. 80
MO/C8 320 320 640 160 320
N.D. = not detected -
* = dead
Example 9.
icles
Micelles according to Example 2 was mixed with formalin killed rota virus part
and subsequently administrated to female mice. After three immunizations the
mice were
made pregnant whereafter the new-born mice were challenged nasally with live
rota virus.
The figures indicate the animals that acquired protection after challenge as
compared to the
total number of animals in that group. The result from this challenge is seen
in Table 8.

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Table 8.
Protection after challenge of rota virus to baby mice where the mother was
vaccinated
with a lipid formulation according to the invention.
Group Administration Protection
Saline im/im/im 2/8
Micelles im/im/im 4/4
Micelles im/nas/nas 6/7
As can be seen from the results there is a good protection both after three
intramuscular
administrations as well as after a primary intramuscular immunization followed
by two
nasal administrations.
Example 10.
To evaluate the toxicity of the lipid formulations these were administrated
into the rat
nasal cavity whereafter the rats were killed and the nasal mucosa were
prepared for light,
fluorescence as well as scanning electron microscopy (SEM). Formulations
according to
Example 1 and Example 2 were tested. Only the mono-olein/pluronic suspension
showed
minor changes in the mucosal surface using the SEM. No effects could be
detected under
light or fluorescence microscopy. The micelles containing mono-olein and oleic
acid were
unable to provoke any changes in the mucosal membranes.
Example 11.
Caco-2 cells, which are a human cell line originating from a colon cancer can
be made
to grow as a epithelial mono layer. These cells are frequently used to examine
different
substances ability to influence the transport of biological substances through
epithelial cells
and has in a number of experimental systems been shown to give a good
correlation to in
vivo data regarding uptake from the gut into the .bloodstream. As marker
substances for
transport through the cells Na-flouresceine or mannitol is used. The
experiments with the
lipid formulations according to this invention showed an enhanced transport
through the
Caco-2 cells at non-toxic concentrations.